The long-term goal of this project is to identify specific inhibitors of B. anthracis helicase and primase and develop them into novel antibiotics for bio-defense. New antibiotics based on novel chemical scaffolds are vital to the bio-defense armory because they are likely to be effective against both natural and engineered resistant forms of bio-terrorist agents and because there are no pre-existing resistance mechanisms. The strategy employed in this project is to screen new targets in well-validated pathways with a diversity of synthetic and natural product compounds to identify new antibiotic structures. 2 key components of the essential DMA replication pathway, helicase (dnaB) and primase (dnaG), which act early and catalyze a rate-limiting step in replication, have not been explored for discovery of bio-defense agents. In all bacterial species studied, these 2 essential enzymes function together as a complex to initiate the polymerization step of DMA replication. These target genes will be cloned, over-expressed, and their essentiality in B. anthracis confirmed. Active proteins will be purified and used to develop an innovative coupled helicase-primase high through put assay based on fluorescence resonance energy transfer (FRET). This screen will be applied to a diverse library of >100,000 discrete small molecule compounds and purified natural products for inhibitors. The novel aspect of this screening assay is its ability to detect inhibition of each of the 2 coupled reactions simultaneously, resulting in immediate deconvolution of the assay results and elimination of many nonspecific hits. Screening hits will be confirmed and any remaining non-specific DNA-binding compounds eliminated by rapid secondary assays for inhibition of a third replication function (DMA polymerase IMC). Compounds will be tested for specificity for DNA replication, and characterized by their mechanism of inhibition. Finally, compounds will be tested for lack of toxicity to mammalian cells in culture, and lack of activity against eukaryotic polymerase alpha and helicase, resulting in a collection of validated hits.